The Rewards and Challenges of Array-Based Karyotyping for Clinical Oncology Applications

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The rewards and challenges of array-based karyotyping for clinical oncology applications

Leukemia (2009) 23, 829–833; doi:10.1038/leu.2009.24 the arrays used for karyotyping include SOMA (SNP oligonucleotide microarrays)7 and CMA (chromosome microarray).8,9 Some consider all platforms to be a type of array comparative Array-based karyotyping is a powerful new technique for genomic hybridization (arrayCGH), whereas others reserve that assessing chromosomal copy number changes that provides term for two-dye methods, and still others who segregate SNP information not previously obtainable by fluorescent in situ arrays because they generate more and different information hybridization (FISH) or conventional cytogeneticsFwhich can than two-dye arrayCGH methods. be both a blessing and a challenge. In this issue, Gunn et al.1 Regardless of the name of the assay or the probe types used, present atypical 11q deletions identified by array-based array-based karyotyping is becoming standard of care for many karyotyping of chronic lymphocytic leukemia (CLL) that may genetic applications and is now on the verge of bursting into be missed by FISH panels used for prognostic stratification of clinical oncology. CLL is an ideal neoplasm to study with copy this disease. Array-based karyotyping is gaining acceptance as a number arrays because (1) the genetic lesions with known clinical tool, and physicians should be prepared to judiciously clinical relevance are chromosomal gains and losses rather interpret results from these platforms. The advantages of array- than balanced translocations and inversions, (2) DNA from a based karyotyping are many and vary somewhat depending on fresh sample is generally available making the analysis more what kind of array is used, but they include high-resolution, straightforward than that for DNA obtained from formalin-fixed genome-wide copy number assessment in one assay; a paraffin-embedded tissue, (3) the tumor burden is known from permanent, numeric result that does not fade over time like the flow cytometry results and can help guide downstream the fluorescent signals of FISH; the ability to karyotype formalin- analysis, (4) the tumor burden tends to be relatively high in the fixed paraffin-embedded tissues and the simultaneous capture of peripheral blood and (5) enrichment for B cells or CLL cells is loss-of-heterozygosity (LOH) status if using a single-nucleotide simple, cost effective and amenable to routine clinical use, polymorphism (SNP)-based array. However, because arrays can which minimizes the effect of ‘normal clone contamination.’ display the genome at high resolution, it is becoming apparent Several groups have recently published manuscripts using copy that the individual molecular lesions identified earlier by FISH number or SNP arrays to study CLL or to validate them for are actually heterogeneous in both genomic length and copy clinical use,5,10–13 and the technique has been successfully number. The clinical meaning of these different subtypes of applied to several solid2–4,14,15 and liquid16–19 tumors. genetic lesions, if any, is yet to be determined. In addition, many In CLL, it is typical to perform a standard FISH panel to genomic changes of uncertain clinical significance are identified determine copy number at key regions of the genome with by these platforms, and there are no standards for reporting such well-established clinical significance, including 6q, 11q, chro- lesions or for archiving them, so that the reports can be amended mosome 12, 13q14 and 17p. In this issue, Gunn et al.1 use a as our knowledge of these lesions evolve. clinically validated array customized to interrogate all known Array-based karyotyping can be carried out with several CLL prognostic loci (179 probes) and 914 FISH-mapped linearly different platforms, both laboratory developed and commercial. distributed clones for whole-genome coverage at an average The arrays themselves can be genome-wide with probes resolution of approximately 2.5 Mb. Their manuscript highlights distributed over the entire genome, or targeted with probes for four atypical 11q deletions. These 11q lesions were considered genomic regions known to be involved in a specific disease or atypical because they did not include the ATM gene, and two of group of diseases, or a combination of both. Furthermore, array- the deletions were missed by the commercial FISH probe used based karyotyping can be carried out with ‘copy number only’ for this locus (11q22.3 Vysis LSI ATM probe, Abbott Molecular, arrays or SNP arrays, which can provide both copy number and Des Plaines, IL, USA). The loss of the ATM tumor suppressor LOH status. The probe types used for ‘copy number only’ arrays gene (TSG), located at 11q22, is often implicated in the include cDNA, BAC clones and oligonucleotides (for example, pathogenesis of CLL. However, it is unlikely to be the sole Agilent (Santa Clara, CA, USA), Nimblegen (Madison, WI, cause of the 11q abnormality, as the minimally deleted region of USA). Commercially available SNP arrays can be solid phase 11q houses other potential candidate TSGs, such as RDX and (Affymetrix, Santa Clara, CA, USA) or bead-based (Illumina, San FDX1 genes,20 and not all CLL patients with deletions of 11q Diego, CA, USA). Some arrays may contain both polymorphic have evidence of an ATM mutation of the remaining allele.21 (SNP-containing) and non-polymorphic (copy number only) Other groups have reported data suggesting that there is a probes, such as the Affymetrix SNP 6.0 array. The actual slightly more telomeric but overlapping region that does not resolution of the virtual karyotype will depend primarily on the include ATM.21–23 This finding was corroborated by Lehmann probe density, the probe performance, the quality of the DNA et al.5 who also observed a second commonly deleted region at and the analysis software. Despite the diversity of platforms, 11q that is telomeric to the ATM gene when they used SNP ultimately they all use genomic DNA from disrupted cells to arrays to karyotype CLL samples. It is postulated that this region recreate a high-resolution karyotype in silico. The end product may harbor another TSG associated with the development of does not yet have a consistent name and has been called virtual CLL, and that concurrent deletion of ATM and this other TSG karyotyping,2,3 digital karyotyping,4 molecular allelokaryotyp- may contribute to a poor prognosis. It is therefore likely that a ing5 and molecular karyotyping.6 Other terms used to describe single FISH probe cannot capture all clinically relevant lesions Editorial 830 at this locus. This article underscores how copy number arrays not only allow us to detect lesions missed by FISH, but also enable us to further refine the break points of 11q deletions and help to determine the clinical relevance, if any, of the subtypes of 11q lesions. Aside from variation in the genomic length of the 11q deletions described by Gunn et al.,1 other types of genetic heterogeneity at clinically relevant loci in CLL have been elucidated by array- based karyotyping. Sargent et al.10 report clinical validation data for a custom CLL oligonucleotide array and highlights length heterogeneity at the 13q14 locus. Patel et al.24 report their clinical validation of a custom CLL BAC array and also underscore the length heterogeneity seen at the 13q14 locus. Ouillette et al.25 used 50 K Affymetrix SNP arrays to subtype 13q14 lesions based on copy number (bi- or mono-alleleic loss) and break points, and they propose a subclassification of 13q14 lesions based on length and gene dose heterogeneity of this locus. Although the lesions detected by FISH have generally been thought of as monolithic, most physicians can readily con- ceptualize length heterogeneity of these deletions. However, Figure 1 Cellular basis of regional heterogeneity seen with array- karyotyping with high resolution using arrays is bringing less based karyotyping. Heterozygous deletions are light blue and easily conceptualized genetic lesions into play, such as copy homozygous deletions are dark blue as depicted within a chromosome neutral LOH (acquired uniparental disomy (UPD)) and regional pair in each cell. (a) Clonal evolution, (b) two separate clones, both scenarios will result in apparent regional copy number heterogeneity copy number heterogeneity. UPD refers to a chromosomal when subjected to array-based karyotyping. region in which both copies of that region are acquired from the same parent, resulting in a copy number of two but with LOH (thus, copy neutral LOH). This can occur through mitotic recombination, chromosome non-dysjunction and loss of one parental chromosome with duplication of the other. Copy neutral LOH can act as the ‘second hit’ of the Knudson two hit hypothesis of tumorigenesis, similar to a deletion, resulting in the removal of the remaining wild-type allele of a TSG.26 Copy neutral LOH is reported to constitute 20–80% of the LOH seen in human cancers, both solid and liquid.16,27–30 Using Affymetrix SNP arrays, Pfeifer et al.12 and Lehmann et al.5 identified copy neutral LOH at clinically relevant loci in CLL. This is noteworthy because conventional cytogenetics, FISH or ‘copy number only’ arrays cannot detect this type of lesion. Array-based karyotyping can also readily detect heterogeneity of copy number state and gene dosage within a particular chromosomal regionFso called ‘regional copy number heterogeneity.’ For example, a region of 13q14 can be deleted in both chromosomes (homozygous deletion), whereas an adjacent, intervening or overlapping genomic region is deleted in only Figure 2 13q14 regional copy number heterogeneity and efficacy of one chromosome (heterozygous deletion). Figure 1a depicts B-cell enrichment. (a) Single-nucleotide polymorphism (SNP) array- clonal evolution of a disease-associated locus, such as 13q14, based karyotype of the 13q14 locus of a chronic lymphocytic showing a single chromosome pair in each cell. In the leukemia (CLL) sample with 17% CD5 þ /CD19 þ CLL cells by flow cytometry. (b) Array-based karyotype of the same sample as (a), but unevolved cells, there is a small, heterozygous deletion (light processed with a density-based B-cell enrichment step before DNA blue) involving only a few genes and underlying the FISH probe extraction revealing a 13q14 aberration that appears partially site. The cells that have undergone clonal evolution have lost a heterozygously deleted (light blue) and partially homozygously larger region of their other chromosome (heterozygous deletion, deleted (dark blue). HMM, Hidden Markov Model. Dark blue light blue) encompassing dozens of genes, which overlaps the indicates copy number of zero, light blue indicates copy number of original heterozygous deletion and converts it into a region of one and yellow indicates copy number of two. The log2 ratio plot is shown as a smoothed average over 10 SNPs. homozygous deletion. Although FISH can show the presence of a mixed population of heterozygously deleted cells (unevolved population) and homozygously deleted cells (cells that have undergone clonal evolution), it cannot determine the gene was generated using DNA from peripheral blood, Affymetrix dosage effects acquired in the original deletion or during the 250 K Nsp SNP array and CNAG3.0 analysis software.31 The clonal evolution. By defining the break points of each lesion, dark blue bar in the Hidden Markov Model indicates a region of array-based karyotyping can identify, for example, that an homozygous deletion (centromeric end) and the light blue band evolved clone has lost both copies of SETDB2, RCBTB1 and indicates a region of heterozygous deletion (telomeric end) ARL11, while still maintaining one copy of DLEU1, DLEU2, within the same genetic lesion at 13q14. FISH for this sample mir-16-1 and mir-15a. An example of a virtual karyotype showed a heterozygous 13q14 deletion in 13% and homo- showing regional copy number heterogeneity of the 13q14 locus zygous deletion in 43% of interphase cells, and most likely in CLL is shown in Figure 2 (unpublished data). The karyotype represents clonal evolution. However, regional copy number

Leukemia Editorial 831 heterogeneity can be due to either clonal evolution or the this sample, 90% of the B cells were CLL cells, which is often the existence of two separate clones in the sample (Figure 2b), and case in CLL. We recommend that samples with less than 30% neither FISH nor array-based karyotyping can distinguish CLL cells should either be enriched before DNA extraction or between the two possibilities. Regional copy number hetero- triaged directly to FISH. Additionally, flow cytometry sorting geneity has been reported by others5,13 and it can be either may be needed for hematopoietic neoplasms with multilineage terminal, as in this example, or interstitial. The ability to detect involvement such as myelodysplastic syndromes. We have the genetic heterogeneity illuminated by array-based karyo- recently shown that multiple distinct clones may co-exist in typing is exciting, but the clinical relevance of this different lineages in myelodysplastic syndromes by using SNP heterogeneityFin genomic length, UPD or regional copy arrays to study the flow sorted marrow samples.33 numberFhas yet to be vetted. As expected, the genome-wide copy number arrays not only This new way of assessing copy number raises the question, see the loci interrogated by the FISH probes, but also see the rest ‘What is the gold standard for copy number detection?’ Each of the genome. Subsequently, genetic lesions of ‘uncertain technique has inherent strengths and weaknesses, and in many clinical significance’ as well as copy number polymorphisms are instances they complement rather than replace one another. often detectedFwhich can be both a blessing and a curse. The Conventional cytogenetics has a very coarse resolution but can higher the density of the array used, the greater the number of identify a multitude of structural lesions, if they are large enough lesions of uncertain significance/copy number polymorphisms and there is fresh tissue available. Copy number arrays can be that will be detected. In a research setting, this can be a rich used on fresh or formalin-fixed paraffin-embedded samples, but source of data for an eager graduate student (or it can be like cannot detect balanced translocation, inversions or lesions in drinking from a fire hose). However, as copy number arrays regions of the genome not represented on the array. In addition, move into routine clinical use for CLL and other tumors, the many copy number analysis software programs used to generate medical professionals performing these assays and signing out array-based karyotypes will falter with less than 25–30% tumor the reports will have to decide what ‘lesions’ will be included in cells in the sample. However, this limitation can be minimized the final report, and the ordering physicians will have to decide by tumor enrichment strategies and/or software optimized for what to do, if anything, with such information. At this time, there use with oncology samples. The analysis algorithms are evolving is no consensus about what ‘lesions’ to include in a clinical rapidly, and some are even designed to thrive on ‘normal clone report for oncology samples studied by copy number arrays. contamination’31 so it is anticipated that this limitation will Some may advocate reporting only those lesions with well- continue to dissipate. FISH, on the other hand, has a reported established clinical relevance, whereas others may advocate sensitivity of approximately 5–7%,10 but can assess only the listing all genetic aberrations identified in the sample. In CLL, specific region of the genome for which it is targeted and it listing the clinically relevant lesions in the diagnostic line and cannot provide information about length of the deletion/ the others in the body of the report is a reasonable approach. duplication. In addition, it is not uncommon for the arrays to This strategy becomes less tenable when reporting on paraffin- detect copy number changes in regions that FISH called normal/ embedded solid tumors, which will have many more genetic diploid because the lesions fell completely or partially outside lesions and more false-positive ‘chatter’Fthe reports could of the region covered by the FISH probe.5,24,25 Whether become quite unwieldy. However, it is imperative for labora- array-based karyotyping will be carried out in lieu of FISH or tories to annotate and archive all genomic aberrations identified conventional cytogenetics remains to be seen, and will most by the arrays, whether or not they are included in the final likely have to be determined on a tumor-by-tumor or case-by- report. This is critical not only for the purpose of novel case basis. biomarker research, but also to be able to readily recall specific The results of the studies using array-based karyotyping to lesions, as our knowledge about them advances from ‘uncertain evaluate CLL have consistently reported high concordance with clinical significance’ to association or lack thereof with FISH panel results, and instances of non-concordance were prognosis, diagnosis, or response to therapy, and to issue explained by low tumor burden (o25–30% CLL cells in the amended reports as indicated. At the time of this writing, the sample), the presence of small subclones, the relatively low commercial tools available for this purpose are scarce and quite resolution of the arrays used in the study and/or differences in primitive. the populations of cells used for each assay. As alluded to Lastly, each type of array and analysis algorithm has inherent above, the presence of normal cells admixed with the tumor strengths and weakness, and the result obtained can be different population will dilute the signal from the tumor. Without tumor depending on such variables as the array density, probe type enrichment steps, genotyping and copy number algorithms will and assumptions underlying the algorithms. These types of fail in the face of ‘normal clone contamination.’ The exact point considerations are unfamiliar territory for most oncologist and of failure, in terms of the minimal percentage of neoplastic cells, pathologists. For example, UPD of key genomic regions in CLL will depend on the particular platform and algorithms used. has been reported at 13q145,12 and 17p,5 and a SNP-based array Enrichment strategies for CLL include FACS cell sorting of can detect UPD, whereas arrayCGH cannot. So, one must CD5 þ /CD19 þ cells,32 magnetic bead separation and density consider whether a negative result at a given locus is negative separation of B cells24 (Stem Cell Technologies, Vancouver, BC, because there is no deletion/UPD present or because the USA). In our hands, enrichment for B cells not only evens the laboratory used an array that cannot detect UPD. But, be careful playing field with regard to comparing log2 ratios between what you wish for. If you detect UPD at a clinically relevant samples, but also resolves lesions in samples with low tumor locus, there are more questions to answer. Acquired UPD may burden or small subclones. Figure 2 shows virtual karyotypes of represent two copies of a mutated TSG or it could represent two chromosome 13 from a sample with 17% CD5 þ /CD19 þ cells unmutated copies. Additional testing, such as sequence by flow cytometry. In the unenriched sample (top), the 13q14 analysis, would have to be carried out to definitively answer deletion was barely discernable and not called by the Hidden that question. Markov Model or segment reporting tools. After density-based Importantly, clinicians need to be aware of the strengths and B-cell enrichment, the lesion is readily evident to the automated limitations of the different types of copy number arrays being calling tools of the software and to the eye of the observer. In used clinically. Although a platform performance comparison

Leukemia Editorial 832 using CLL samples has been published,34 conclusions about 11 Schwaenen C, Nessling M, Wessendorf S, Salvi T, Wrobel G, algorithms are often out of date by the time they go to press and Radlwimmer B et al. Automated array-based genomic profiling in new arrays are continually on the market. In this rapidly chronic lymphocytic leukemia: development of a clinical tool and evolving field, both the arrays and the analysis software discovery of recurrent genomic alterations. Proc Natl Acad Sci USA 2004; 101: 1039–1044. continue to mature. There are currently several platforms that 12 Pfeifer D, Pantic M, Skatulla I, Rawluk J, Kreutz C, Martens UM have been clinically validated and are amenable to routine et al. Genome-wide analysis of DNA copy number changes and clinical use for detecting copy number alterations in CLL. The LOH in CLL using high-density SNP arrays. Blood 2007; 109: complexities of in silico karyotyping may fluster many 1202–1210. physicians, but array-based karyotyping is most likely to be an 13 Gunn SR, Mohammed MS, Gorre ME, Cotter PD, Kim J, increasingly used tool in CLL and other neoplasms. It is worth Bahler DW et al. Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in the effort to familiarize oneself with these platforms. These chronic lymphocytic leukemia. J Mol Diagn 2008; 10: 442–451. arrays are a seemingly endless source of novel potential 14 Haverty PM, Fridlyand J, Li L, Getz G, Beroukhim R, Lohr S et al. biomarkers to investigate and are inching us ever closer to High-resolution genomic and expression analyses of copy number personalize medicine. alterations in breast tumors. Genes Chromosomes Cancer 2008; 47: 530–542. JM Hagenkord1 and CC Chang2,3 15 Kurashina K, Yamashita Y, Ueno T, Koinuma K, Ohashi J, Horie H 1Creighton Medical Laboratories and Department of et al. Chromosome copy number analysis in screening for Pathology, Creighton University Medical Center, Omaha, prognosis-related genomic regions in colorectal carcinoma. NE, USA; Cancer Sci 2008; 99: 1835–1840. 2Department of Pathology, The Methodist Hospital and 16 Gondek LP, Tiu R, O’Keefe CL, Sekeres MA, Theil KS, Maciejewski The Methodist Hospital Research Institute, Houston, JP. Chromosomal lesions and uniparental disomy detected by SNP TX, USA and arrays in MDS, MDS/MPD, and MDS-derived AML. Blood 2008; 3Department of Pathology, Weill Medical College of Cornell 111: 1534–1542. University, New York, NY, USA 17 Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J, Ma J et al. E-mail: [email protected] BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature 2008; 453: 110–114. 18 Mullighan CG, Phillips LA, Su X, Ma J, Miller CB, Shurtleff SA et al. Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia. Science 2008; 322: 1377–1380. References 19 Huang WT, Yang X, Zhou X, Monzon FA, Wen J, Hagenkord JM et al. Multiple distinct clones may co-exist in different lineages 1 Gunn S, Hibbard M, Ismail S, Lowery-Nordberg M, Mellink C, in myelodysplastic syndromes. Leuk Res 2008; [e-pub ahead of print]. Bahler D et al. Atypical 11q deletions identified by array CGH may 20 Stilgenbauer S, Liebisch P, James MR, Schroder M, Schlegelberger be missed by FISH panels for prognostic markers in chronic B, Fischer K et al. Molecular cytogenetic delineation of a novel lymphocytic leukemia. Leukemia 2009; 23: 1011–1017. critical genomic region in chromosome bands 11q22.3-923.1 in 2 Hagenkord JM, Parwani AV, Lyons-Weiler MA, Alvarez K, lymphoproliferative disorders. Proc Natl Acad Sci USA 1996; 93: Amato R, Gatalica Z et al. Virtual karyotyping with SNP 11837–11841. microarrays reduces uncertainty in the diagnosis of renal epithelial 21 Cotter FE, Auer RL. Genetic alteration associated with chronic tumors. Diagn Pathol 2008; 3: 44. lymphocytic leukemia. Cytogenet Genome Res 2007; 118: 310–319. 3 Monzon FA, Hagenkord JM, Lyons-Weiler MA, Balani JP, 22 Zhu Y, Monni O, El-Rifai W, Siitonen SM, Vilpo L, Vilpo J et al. Parwani AV, Sciulli CM et al. Whole genome SNP arrays as a Discontinuous deletions at 11q23 in B cell chronic lymphocytic potential diagnostic tool for the detection of characteristic leukemia. Leukemia 1999; 13: 708–712. chromosomal aberrations in renal epithelial tumors. Mod Pathol 23 Auer RL, Jones C, Mullenbach RA, Syndercombe-Court D, 2008; 21: 599–608. Milligan DW, Fegan CD et al. Role for CCG-trinucleotide repeats 4 Leary RJ, Lin JC, Cummins J, Boca S, Wood LD, Parsons DW et al. in the pathogenesis of chronic lymphocytic leukemia. Blood 2001; Integrated analysis of homozygous deletions, focal amplifications, 97: 509–515. and sequence alterations in breast and colorectal cancers. Proc 24 Patel A, Kang SH, Lennon PA, Li YF, Rao PN, Abruzzo L et al. Natl Acad Sci USA 2008; 105: 16224–16229. Validation of a targeted DNA microarray for the clinical evaluation 5 Lehmann S, Ogawa S, Raynaud SD, Sanada M, Nannya Y, of recurrent abnormalities in chronic lymphocytic leukemia. Am J Ticchioni M et al. Molecular allelokaryotyping of early-stage, Hematol 2008; 83: 540–546. untreated chronic lymphocytic leukemia. Cancer 2008; 112: 25 Ouillette P, Erba H, Kujawski L, Kaminski M, Shedden K, Malek 1296–1305. SN. Integrated genomic profiling of chronic lymphocytic leukemia 6 Vermeesch JR, Fiegler H, de Leeuw N, Szuhai K, Schoumans J, identifies subtypes of deletion 13q14. Cancer Res 2008; 68: Ciccone R et al. Guidelines for molecular karyotyping in consti- 1012–1021. tutional genetic diagnosis. Eur J Hum Genet 2007; 15: 1105–1114. 26 Mao X, Young BD, Lu YJ. The application of single nucleotide 7 Kulharya AS, Flannery DB, Norris K, Lovell C, Levy B, polymorphism microarrays in cancer research. Curr Genomics Velagaleti GV. Fine mapping of breakpoints in two unrelated 2007; 8: 219–228. patients with rare overlapping interstitial deletions of 9q with mild 27 Beroukhim R, Lin M, Park Y, Hao K, Zhao X, Garraway LA et al. dysmorphic features. Am J Med Genet 2008; 146A: 2234–2241. Inferring loss-of-heterozygosity from unpaired tumors using high- 8 Nowakowska B, Stankiewicz P, Obersztyn E, Ou Z, Li J, density oligonucleotide SNP arrays. PLoS Comput Biol 2006; Chinault AC et al. Application of metaphase HR-CGH and targeted 2: e41. Chromosomal Microarray Analyses to genomic characterization of 28 Huang J, Wei W, Zhang J, Liu G, Bignell GR, Stratton MR et al. 116 patients with mental retardation and dysmorphic features. Am Whole genome DNA copy number changes identified by high J Med Genet 2008; 146A: 2361–2369. density oligonucleotide arrays. Hum Genomics 2004; 1: 287–299. 9 Probst FJ, Roeder ER, Enciso VB, Ou Z, Cooper ML, Eng P et al. 29 Ishikawa S, Komura D, Tsuji S, Nishimura K, Yamamoto S, Chromosomal microarray analysis (CMA) detects a large X Panda B et al. Allelic dosage analysis with genotyping microarrays. chromosome deletion including FMR1, FMR2, and IDS in a female Biochem Biophys Res Commun 2005; 333: 1309–1314. patient with mental retardation. Am J Med Genet 2007; 143A: 30 Lo KC, Bailey D, Burkhardt T, Gardina P, Turpaz Y, Cowell JK. 1358–1365. Comprehensive analysis of loss of heterozygosity events in 10 Sargent R, Jones D, Abruzzo LV, Yao H, Bonderover J, Cisneros M glioblastoma using the 100K SNP mapping arrays and comparison et al. Customized oligonucleotide array-based comparative with copy number abnormalities defined by BAC array compara- genomic hybridization as a clinical assay for genomic profiling tive genomic hybridization. Genes Chromosomes Cancer 2008; of chronic lymphocytic leukemia. J Mol Diagn 2009; 11: 25–34. 47: 221–237.

Leukemia Editorial 833 31 Yamamoto G, Nannya Y, Kato M, Sanada M, Levine RL, Kawamata 33 Huang WT, Yang X, Zhou X, Monzon FA, Wen J, Hagenkord JM N et al. Highly sensitive method for genomewide detection of et al. Multiple distinct clones may co-exist in different lineages allelic composition in nonpaired, primary tumor specimens by use in myelodysplastic syndromes. Leuk Res 2008; e-pub ahead of affymetrix single-nucleotide-polymorphism genotyping micro- of print. arrays. Am J Hum Genet 2007; 81: 114–126. 34 Gunnarsson R, Staaf J, Jansson M, Ottesen AM, Goransson H, 32 Kujawski L, Ouillette P, Erba H, Saddler C, Jakubowiak A, Liljedahl U et al. Screening for copy-number alterations and loss of Kaminski M et al. Genomic complexity identiﬁes patients with heterozygosity in chronic lymphocytic leukemiaFa comparative aggressive chronic lymphocytic leukemia. Blood 2008; 112: study of four differently designed, high resolution microarray 1993–2002. platforms. Genes Chromosomes Cancer 2008; 47: 697–711.

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